Artemisinin (AN), produced by the plant Artemisia annua L. and delivered as ACT (Artemisinin Combination Therapy), is a proven sesquiterpene lactone therapeutic for treating malaria and shows promise against many other diseases. This important medicinal herb is also becoming of interest to the N. American market, particularly to promote general health and healing via its anti-inflammatory effects. Recently we reported that whole plant A. annua (pACT) is at least fivefold more effective against parasitemia, >40-fold more bioavailable, and much more resilient to development of drug resistance than pure AN. Together these results suggest the native chemical complexity of pACT is facilitating these responses. Some endogenous small molecules produced by the plant have shown antiplasmodial activity; some also show therapeutic synergism with AN vs. falciparum malaria. However, little is known about the effect of these assistive compounds (ACs), mainly flavonoids and terpenes, on bioavailability of AN. Their passage through the intestinal wall into the serum is also not clear. Our fundamental studies will help explain how the plant matrix and its endogenous small molecules affect bioavailability of AN, important to improving overall understanding of pACT efficacy. Aim 1 The target ACs, verified to be present in the A. annua cultivar (SAM) used in pACT, may enhance bioavailability of AN as observed in our animal studies. To determine if the ACs enhance transport through the intestinal wall, we will use the in vitro digestion model, Caco-2, with pure AN + combinations of the pure test ACs to measure AN and AC permeability. This will tell us how the pure ACs affect AN intestinal passage and indicate if any of the ACs also pass through the intestinal wall, but absent the background plant matrix. Aim 2 To determine how the plant matrix of pACT affects bioavailability of AN, we will measure the Caco-2 permeability of AN from ex vitro intestinal digestion of the pACT plant matrix produced from SAM (contains ACs). We will also measure the level of passaged ACs. This tells us how much AN and ACs move from the apical to basolateral side of the Caco-2 systems and becomes our comparative control against which results of Aim 3 will be measured. Aim 3 Using intestinal digestate of the GLS plant matrix we will systematically add AN along with individual ACs known to be absent (or nearly so) in GLS, but present in SAM (the pACT plant), to determine which is effective in improving the passage of AN. This will tell us which of the ACs is effective in assisting the passage of AN, but with a null plant matrix. Aim 4 Using serum already on hand from pACT-fed mice, we will also extract and measure ACs for their presence in the bloodstream. We already have on hand frozen serum extracts from our recent PK experiment (see Sec. 3.3.3). While we have now measured AN in those extracts, we have waited until we have accumulated relavant AC standards to quantify their levels in these serum samples. This will tell us if any ACs actually appears in the serum.